An Antisense Oligonucleotide Is the First Drug to Demonstrate Reduction of Mutant Huntingtin Protein in Humans

  • Mutant Huntingtin Protein Causes Huntington Disease (HD), Which Afflicts 30,000 People in the U.S.
  • More Than 200,000 People at Risk of Inheriting HD in the U.S.
  • Developed by Ionis Pharmaceuticals, This Antisense Drug Will Undergo Pivotal Clinical Trials Conducted by Roche

Credit: A Luna Blue

My blog from August 7th, 2018, heralded the clinical efficacy of two oligonucleotide drugs for transthyretin-related amyloidosis. One is an antisense oligonucleotide (ASO) drug, and the other is a short-interfering RNA drug. Five days before this notable achievement, Ionis Pharmaceuticals announced equally important news, stating that the European Medicines Agency granted accelerated review timelines for an ASO (IONIS-HTTRx) for the treatment of people with Huntington’s disease (HD), a neurodegenerative illness.

IONIS-HTTRx is the first drug to demonstrate reduction of mutant huntingtin protein, the underlying cause of HD, which is the focus of the present blog. It should be noted that Ionis and Roche have a long-standing alliance when it comes to HD, under which IONIS-HTTRx (designated RG6042 by Roche) will be evaluated in a pivotal study of a larger patient population to further characterize its safety and efficacy profile in adults with HD.

Basic Facts About HD

Description: According to the NIH, HD (aka Huntington’s disease or Huntington’s chorea) is a progressive brain disorder that causes uncontrolled movements, emotional problems, and loss of thinking ability (cognition). Adult-onset HD, the most common form of this disorder, usually appears in a person’s 30s or 40s. Early signs and symptoms can include irritability, depression, small involuntary movements, poor coordination, and trouble learning new information or making decisions. Affected individuals may have trouble walking, speaking, and swallowing. People with HD also experience changes in personality and a decline in thinking and reasoning abilities. Individuals with the adult-onset form of HD usually live about 15 to 20 years after signs and symptoms begin.

A less common form of HD known as the juvenile form begins in childhood or adolescence. It also involves movement problems and mental and emotional changes. Additional signs of the juvenile form include slow movements, clumsiness, frequent falling, rigidity, slurred speech, and drooling. Juvenile HD tends to progress more quickly than the adult-onset form; affected individuals usually live 10 to 15 years after signs and symptoms appear.

Frequency: HD affects an estimated 3 to 7 per 100,000 people of European ancestry. The disorder appears to be less common in some other populations, including people of Japanese, Chinese, and African descent.

Causes: As depicted here, mutations in the HD gene/HTT gene on chromosome 4 cause Huntington disease. The HTT gene provides instructions for making a protein called huntingtin. Although the function of this protein is unknown, it appears to play an important role in nerve cells (neurons) in the brain, primarily a group of nerve cells at the base of the brain known collectively as the basal ganglia.

Credit: Meletios Verras

The HTT mutation that causes HD involves a DNA segment known as a CAG trinucleotide repeat. Normally, the CAG segment is repeated 10 to 35 times within the gene. In people with HD, the CAG segment is repeated 36 to more than 120 times. People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with 40 or more repeats almost always develop the disorder.

Neuron structure

CAG codes for glutamine, therefore an increase in the size of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. The dysfunction and eventual death of neurons in certain areas of the brain underlie the signs and symptoms of HD.

Inheritance: As depicted here, HD is inherited in an autosomal dominant pattern wherein one copy of the altered gene in each cell is sufficient to cause the disorder. An affected person usually inherits the altered gene from one affected parent. In rare cases, an individual with HD does not have a parent with the disorder.

As the altered HTT gene is passed from one generation to the next, the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. People with the adult-onset form of HD typically have 40 to 50 CAG repeats in the HTT gene, while people with the juvenile form of the disorder tend to have more than 60 CAG repeats.

Individuals who have 27 to 35 CAG repeats in the HTT gene do not develop Huntington disease, but they are at risk of having children who will develop the disorder. As the gene is passed from parent to child, the size of the CAG trinucleotide repeat may lengthen into the range associated with HD (36 repeats or more).

Genetic Testing: There are 64 available genetic tests for HD listed at a NIH website that you can access here. Among these 64, I am most interested in the 7 tests described as “sequence analysis of the entire coding region,” which to me seem to be the most definitive approach. Of these 7 tests, I clicked on this link for the test named “HTT,” which was the only one of these tests offered by a U.S.-based company, namely, Fulgent Genetics near Los Angeles, CA. While this HTT test is described as using “massively parallel sequencing,” which Fulgent specifies as an Illumina® test, this test’s clinical validity and clinical utility are described as “not provided.” I assume this means that this HTT test can be ordered by a doctor for informational purposes. In any case, there is a tab given for “How To Order” that interested readers can consult.

PCR aficionados will recognize HHT’s CAG repeats to be GC-rich, making them difficult to faithfully amplify on Illumina platforms or through other types of ensemble sequencing methods. However, this can be mitigated by using TriLink CleanAmp® 7-deaza-dGTP. Amplification-free methodology is also available. For example, a poster abstract by Pacific Biosystems (PacBio) describes how a novel approach using CRISPR/Cas9 for specific targeting of individual human genes, followed by PacBio’s single-molecule long-read sequencing methods, enables sequencing of complex genomic regions that cannot be investigated with other technologies. HTT CAG repeat-regions were successfully sequencing in this manner.

ASO Studies Targeting Huntington

Use of an ASO to interfere with the expression of mutant HTT can be traced back to 20 years ago, through the following series of publications by various research groups:

Of these, interested readers can consult the last item by Kordasiewicz et al. (2012); a lengthy, detailed report by collaborators at four sites, including Ionis (then named Isis Pharmaceuticals). In brief, this study demonstrated that transient infusion of ASOs into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal.

Rhesus monkey eating

Similar ASO infusion into non-human primates (Rhesus monkeys) was shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, these findings established a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Finally, Kordasiewicz et al. note that huntingtin is reportedly essential for one or more early developmental steps. However, no evidence to date has demonstrated toxicity following suppression of huntingtin in the adult brain. In fact, their ASO-mediated simultaneous suppression of mutant and normal huntingtin by 60% in the adult rodent striatum, and suppression of normal huntingtin by 45% in the non-human primate striatum, were both well tolerated.

Readers who are practiced in synthetic or medicinal chemistry are likely interested in the structure of IONIS-HTTRx. After considerable research, I found a SlideShare on LinkedIn, shown below. The SlideShare shows a 5-10-5 20-base gapmer comprised of ‘Generation 2+’ ASO with all-phosphorothioate- and 2’ MOE-modifications. It should be noted, however, that this is only an exemplary generic structure, as the actual sequence is proprietary, according to Anne Smith, the Director of Clinical Development at Ionis, who I contacted for permission to show this image.

Exemplary generic structure of IONIS-HTTRx. With permission from Anne Smith, Ionis

Before outlining future clinical studies of this ASO in the next section, I’ll conclude this section by providing a link to more than 100 items listed in Google Scholar for “Huntington and TriLink,” which can be perused later, as there are many interesting finds. One exemplary item that caught my attention was a patent for single-domain antibodies, which can be used in therapeutic methods to inhibit huntingtin protein aggregation.

Roche’s Clinical Trials of IONIS-HTTRx Renamed as RG6042

In December 2017, Roche acquired development and marketing rights to RG6042 from Ionis. According to an article from September 24, 2018 in Huntington’s Disease News, two new clinical studies by Roche of IONIS-HTTRx—now RG6042—for HD are planned to start by the end of 2018, and will begin enrolling participants by early 2019. These studies will help researchers understand progression of HD and the therapeutic effectiveness of RG6042, which may ‘potentially be the biggest breakthrough in neurodegenerative disease in the past 25 years,’ according to an interview with C. Frank Bennett, PhD, Ionis’ Senior Vice President of Research and franchise leader for the neurological programs.

The upcoming HD Natural History study and the Phase 3 GENERATION HD1 trial were announced at the recent 2018 European Huntington’s Disease Network plenary meeting in Vienna. The 15-month HD Natural History study will assess the correlation between mutant huntingtin protein in cerebral spinal fluid and in other clinical measures of HD, and will also evaluate wearable devices to measure disease burden. There is no therapeutic treatment in this study, as the goal is to understand the natural progression of the disease. The study will include up to 100 early-stage symptomatic patients at sites in the U.S., U.K., Canada, and Germany, and its results are expected to provide valuable information for the Phase 3 GENERATION HD1 study.

The two-year, global GENERATION HD1 trial will evaluate the long-term safety and effectiveness of RG6042 in up to 660 patients with symptoms of HD. It will be ‘the world’s first Phase 3 study to measure the effect’ of a therapy that lowers the amount of mutant huntingtin protein, according to Bennett. The trial will be conducted at 80 to 90 sites in 15 countries around the world.

2019 Breakthrough Prize for Bennett

On October 17, 2018, The Breakthrough Prize Foundation and its well-known sponsors—Sergey Brin, Priscilla Chan and Mark Zuckerberg, Ma Huateng, Yuri and Julia Milner, and Anne Wojcicki, announced the recipients of the 2019 Breakthrough Prize, awarding a collective total of $22 million to nine researchers for important achievements in the Life Sciences, and in Fundamental Physics and Mathematics. Considered the world’s most generous science prize, each Breakthrough Prize is for $3 million.

C. Frank Bennett. With his permission

By remarkable coincidence, The Breakthrough Prize in Life Sciences this year was jointly awarded to C. Frank Bennett at Ionis, and Adrian R. Krainer at Cold Spring Harbor Laboratory. The citation reads “for the development of an effective antisense oligonucleotide therapy for children with the neurodegenerative disease spinal muscular atrophy.”

Spinal muscular atrophy (SMA) is a rare but devastating disease, and the leading genetic cause of infant death. Many children with SMA die before their second birthday. Now, it is no longer a death sentence. Frank Bennett, a pharmacologist, and Adrian Krainer, a biochemist, built upon their discoveries on antisense technology and the natural process of RNA splicing to produce the first drug to treat SMA—Nusinersen (marketed by Biogen as Spinraza). It was approved by the FDA in 2016.

Those who are interested can learn more about SMA and Nusinersen by reading my October 2016 blog, which discusses this exciting breakthrough.

The work by Bennett on an ASO for treatment of SMA, and now his principal involvement in the development of IONIS-HTTRx as a promising drug for HD, are extraordinary contributions to science and society.

I’m more than pleased to have had the opportunity to collaborate with Frank in the early days of Isis Pharmaceuticals.

As usual, your comments are welcomed.


After writing this blog, I came across some important news regarding the identification of sensitive indicators of HD progression and outcome of therapeutic intervention. Byrne et al. assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF), as well as blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. The concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. These findings were said to provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD, and could be integrated into both clinical trials and the clinic itself.

Long Noncoding RNA (lncRNA) Revisited

  • Publications Dealing with lncRNAs Show Exponential Growth
  • Evidence for Involvement of lncRNAs in Cancer is Increasing
  • Value of lncRNAs as Biomarkers Has Been Validated

Several years ago, I posted a blog about long noncoding RNAs (lncRNAs), which are defined as non-protein coding transcripts in the range of ~200 nt to ~100 kb long. Interest in lncRNA—and other types of noncoding RNA such as microRNA (miRNA) and short interfering RNA (siRNA)—is fueled in large part by a collective scientific desire to uncover and understand the existence and function of all forms of RNA dark matter, so named by analogy to dark energy in cosmology. The lncRNA component of RNA dark matter is certainly generated from transcription of noncoding (formerly “junk”) DNA, but much has yet to be elucidated about function.

As depicted below, lncRNA (red) may act as (a) decoys to release proteins from chromatin, (b) scaffolds for grouping protein complexes, (c) guides to recruit proteins or (d) transcriptional enhancers by bending chromatin. Not shown is lncRNA acting as an antagonist for other regulatory noncoding RNAs, namely miRNA, which can be studied by next-generation sequencing methods such as TriLink’s CleanTag approach.

Taken from Bohla et al. Dis Markers (2017)

Numerical support for upward trending interest in lncRNA is provided by the chart shown here for the number of annual publications on lncRNA. This chart was produced by using data I found in PubMed for 2000-2016, which clearly show a relatively flat rate of ~150 papers per year from 2000-2007, and then an exponential increase to ~2,220 papers in 2016.

Although it’s not possible to say for sure what catalyzed this marked upturn in lncRNA publications, searching the 2005 literature in Google Scholar led to finding the following top-5 cited publications, which from titles alone could be likely scientific co-catalysts:

In any case, when I did keyword searches of the ~13,000 publications on lncRNA, roughly one-third (~4,700) were related to cancer, and many (~1,800) dealt with biomarkers primarily for (~1,400) cancer, but also including cardiovascular diseases, diabetes, epilepsy, general anxiety disorder, inflammatory bowel diseases, etc. Given that cancer is a major medical problem for all countries to deal with, and knowing that early detection of cancer by finding better biomarkers is critically important, this blog revisits lncRNA in the context of cancer and biomarkers.

State-of-the-Art Technologies to Explore lncRNA

At the risk of over simplification, advances in RNA sequencing (RNA-Seq)—enabled largely by high-throughput instrumentation from Illumina, PacBio, and Thermo Scientific—has revolutionized the field of molecular biology by revealing that up to ~75% of the human genome is actively transcribed, and that most of this transcriptome consists of lncRNA. Bioinformatic analyses, which are way beyond my expertise, have played a key role in sorting out lncRNA from mRNA and other RNA species. Interested readers can consult Cobos et al. as a recent lead reference to learn more about these bioinformatic methods.

Following are links to a constellation of additional experimental techniques that are available for exploring lncRN, and can be perused in detail later:

Although these methods are employed to shed light on lncRNA cellular localization, structure, interaction networks and functions, interested readers should consult a review by Salehi et al. and research paper by Goyal et al. for discussions of the advantages and disadvantages of these techniques. For example, Goyal et al. note that many lncRNA are derived from bidirectional promoters, or overlap with promoters, or bodies of sense or antisense genes. In a genome-wide analysis, they found only 38% of 15,929 lncRNA loci are safely amenable to CRISPR applications, while almost two-thirds of lncRNA loci are at risk to inadvertently deregulate neighboring genes. For several representative lncRNAs, it was found that CRISPR—but not RNAi by siRNA or antisense oligos—also affects their respective neighboring genes.

In closing this section on methods, readers who follow my blogs know that I’m a big fan of nanopore sequencing, about which I’ve commented in several previous posts. Oxford Nanopore Technologies has recently announced advances in its nanopore technologies that now allow sequencing of an RNA strand directly, rather than analyzing the products of reverse transcription and PCR reactions. My scientific “crystal ball” sees use of nanopore sequencing of lncRNAs in the not too distant future.

Long Noncoding RNA in Cancer and as Biomarkers

According to Bohla et al., lncRNAs are now known to function as regulatory factors for numerous, important cellular processes, such as growth, differentiation, and cell death. In addition, lncRNAs are involved in controlling alternative splicing, regulation of gene expression at the posttranscriptional level, chromatin modification, inflammatory pathologies, and—when deregulated—various types of cancer. Lnc2Cancer is a manually curated, interactive database of cancer-associated lncRNAs with experimental support that provides a high-quality and integrated resource for exploring lncRNA deregulation in various human cancers. In my opinion, Lnc2Cancer is definitely worth perusing.

The figure shown here indicates various types of cancers (black) for which lncRNAs (names of which are in green, blue or red) have been implicated. Searching PubMed or Google Scholar using any of the lncRNA names will provide a host of publications to peruse.

Taken from Vitiello et al. Cellular Oncology (2015)

For example, searching PubMed for the term “HOTAIR” gives ~475 publications, in chronological order starting with the most recent. By contrast, searching Google Scholar for the terms “HOTAIR lncRNA” gives ~6,100 articles ranked by “relevance,” which is explained elsewhere as being heavily influenced by citation frequency. Readers interested in more details can consult Lin & Yang, who review the mechanisms by which lncRNAs regulate cellular responses to extracellular signals, and discuss the clinical potential of lncRNAs as diagnostic indicators, stratification markers and therapeutic targets of combinatorial treatments.

As was mentioned in the introduction, there are numerous publications aimed at using these and other cancer-associated lncRNAs as biomarkers. Among the main advantages of lncRNAs that make them suitable as cancer diagnostic and prognostic biomarkers, high stability while circulating in body fluids, especially when included in exosomes or apoptotic bodies, is noted by Bohla et al. Despite abundant quantities of ribonucleases in different body fluids, lncRNAs protected in exosomes or apoptotic bodies can be detected in whole blood, plasma, urine, saliva and gastric juice. These lncRNAs as biomarkers are obtainable by non- or minimally invasive methods, which are well tolerated by patients compared to conventional biopsies.

Taken from

Challenges for use of lncRNAs as biomarkers include development of convenient, low cost yet robust isolation methods, and accurate quantitation of relatively low copy numbers, which heretofore has relied on an amplification step, such as enzymatic conversion into cDNA followed by PCR. However, single-molecule detection approaches have evolved to obviate the need for amplification.

For example, NanoString Technologies now offers the nCounter® lncRNA Assay for validation of lncRNA discoveries, which can then be followed by use for biomarker quantification. As depicted below in the left panel, single molecules of lncRNAs (red) can be detected at the same time as mRNAs green and blue), if so desired, using sequence-specific probes each having a fluorescence-based “barcode” identifier. The right panel depicts extension of this approach to identify lncRNA-protein interactions by inclusion of antibody precipitation. This digital-counting assay allows researchers to select up to 800 lncRNAs for analysis in a single multiplexed reaction, which is quite impressive, in my opinion.

Taken from

Taken from Cesano J Immunother Cancer (2015)

Readers interested in more details for application of nCounter® for analysis of biomarkers are referred to a recent publication by Permuth et al. dealing with pancreatic ductal adenocarcinoma (PDAC), which is an aggressive disease that lacks effective biomarkers for early detection. Briefly, these researchers hypothesized that circulating lncRNAs may act as diagnostic markers, and used nCounter® technology to measure the abundance of 28 candidate lncRNAs in pre-operative plasma from a cohort of pathologically-confirmed PDAC cases of various grades of severity and non-diseased controls. Results showed that two lncRNAs aided in differentiating PDAC from controls, and an 8-lncRNA signature had greater accuracy than standard clinical and radiologic features in distinguishing ‘aggressive/malignant’ PDAC that warrant surgical removal from ‘indolent/benign’ PDAC. In my opinion, these findings seem very promising for use with PDAC and, by conceptual extension, to other cancers.

Temozolomide (TMZ). Taken from

As a final example of lncRNA biomarkers for cancer, MALAT1 (pictured above) has been recently reported by Chen et al. as a prognostic factor in glioblastoma multiforme (GMF), and induces chemoresistance to temozolomide (TMZ). The significance of these findings is that GBM is the most malignant brain tumor with limited therapeutic options, and that TMZ is first-line chemotherapy for GBM. These researchers first used deep-sequencing and bioinformatic methods to identify lncRNAs showing different expression levels in TMZ-resistant and non-resistant patients. RT-qPCR was then performed in tissues and serum samples, and lncRNA MALAT1 was shown to discriminate between responding patients from non-responding patients.

Closing Comments

If you’re are interested knowing much more about lncRNAs, then is a great website for you to visit and subscribe to, if you want to keep current on all manner of lncRNA research and industry news. This interactive blog, which posts abstracts and images from the latest lncRNA publications, allows readers to post comments that allow you to join in the “conversation” or simply follow what others are thinking about these articles.

Also provided are links to a host of different online tools for lncRNA research and development, as well as “what’s happening” in terms of upcoming lncRNA events or conferences. Those of you currently seeking a new position may find the jobs postings to be helpful.

Finally, there are there plenty of pop-up advertisements and commercial banners, but these are also informative about lncRNA products and services that are available.

As usual, your comments are welcomed.

Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies


Taken from 

Taken from

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.


In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from

Taken from

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from

Taken from

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from

Taken from

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from

Taken from

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from

Taken from

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Small RNA is Big Science

  • Most Top-5 Citations in Clinical Chemistry are MicroRNA (miRNA) Biomarkers
  • miRNA Biomarker Bonanza is Predicted by Panel of Experts, Although No miRNA Biomarkers Have Yet Been Approved by FDA
  • Plethora of Potential Short Regulatory RNA Exists Beyond the Typical miRNA Microcosm

I’m always looking for new and hopefully engaging topics to comment on, and a recent “Best of Clinical Chemistry” item featured in a special issue of Clinical Chemistry definitely caught my attention. I wasn’t surprised by MIQE Guidelines being at the top, given that these are the “bible” for doing accurate quantitative PCR (qPCR) that has become a seemingly ubiquitous molecular assay for clinical studies. However, I was totally surprised that the next four “best of” all involved microRNA (miRNA)! Hence today’s blog about these small RNA being big science—play on words intended (although properly speaking I should say short rather than small). Continue reading

Pseudouridine Biomarker for Breast Cancer

As you are probably aware, October is National Breast Cancer Awareness month. Everyone from the NFL to Yoplait yogurt seems to be engaged in campaigns for fundraising and awareness. I think it’s great to see the extensive community support for this worthy cause. Since breast cancer awareness is top of mind this month, I thought I’d follow up my latest blog about pseurdouridine with a ‘mini blog’ highlighting some interesting research involving a pseudouridine biomarker for breast cancer.

I’d also like to mention that TriLink is participating in Breast Cancer Awareness month. For every order placed in October, TriLink will dontate $5 to Susan G Komen to support the upcoming 3-Day Walk being held November 20-22 in San Diego.


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Gene-Expression Biomarkers Can Detect Depression

  • First-ever Lab Test for Depression Found Using RT-PCR
  • FDA Approval as Diagnostic Possible by Early 2016
  • Huge Potential Market as 1-in-10 US Adults Suffer from Depression

While it’s normal for everyone to occasionally feel blue or sad, prolonged bouts of depression that interfere with normal life are indicative of a serious mental health issue. While there are numerous forms and differing severity of depressive disorders, as described at a National Institute of Mental Health (NIMH) website, only two factual aspects of this illness really stand out in my opinion:

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

  • Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.
  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.

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Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics

  • Invasive Needles and Scalpels Seen as Passé
  • Noninvasive Sampling Advocates Focusing on Circulating Tumor Cells (CTCs) 
  • New Companies are Pursuing the Liquid Biopsy “Gold Rush”

Biopsy Basics

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from via Bing Images).

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from via Bing Images).

As defined in Wikipedia, a biopsy is ‘a medical test commonly performed by a surgeon or an interventional radiologist involving sampling of cells or tissues for examination.’ Biopsies can be excisional (removal of a lump or area), incisional (removal of only a sample of tissue), or a needle aspiration (tissue or fluid removal). Despite the value of these traditional types of biopsies, they are more or less invasive, lack applicability in certain instances, and require accurately “going to the source” of concern, as pictured to the right, for ultrasound-guided breast cancer biopsy. Better methodology is highly desirable and is the topic of this post. By the way, if you want to peruse a lengthy list of scary risks associated with various type of common invasive biopsies, click here to see what I found in Google Scholar by searching “incidence of complications from biopsies.”

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